Antihemophilic factor prepared from blood plasma using polyethylene glycol

ABSTRACT

ANTIHEMOPHILIC FACTOR IS SEPARATED AS A PRECIPITATE FROM FRESH-FROZEN HUMAN PLASMA WITH A LOW CONCENTRATION OF ETHANOL, PREFERABLE 3 PERCENT, AT LOW TEMPERATURE, FOLLOWED BY EXTRACTION WITH A LOW IONIC STRENGTH BUFFER, PREFERABLY A TRIS BUFFER SOLUTION. THE RESULTING PRODUCT OF INTERMEDIATE PURITY (25-60X PURIFIED) CAN BE LYOPHILIZED AND STORED OR CAN BE FURTHER TREATED TO OBTAIN A HIGH PURITY AHF (200-400X PURIFIED). HIGH PURITY AHF IS PRODUCED BY FURTHER TREATING THE INTERMEDIATE PURITY PRODUCT WITH PEG, PREFERABLY OF A MOLECULAR WEIGHT OF 4,000, IN TWO STEPS, THE FIRST AT ABOUT 6 PERCENT AND THE SECOND AT ABOUT 12 PERCENT. THE HIGH PURITY PRODUCT IS LYOPHILIZED BEFORE STORAGE. BOTH THE INTERMEDIATE, AND THE HIGH, PURITY AHF ARE USED IN THE TREATMENT OF CLASSICAL HEMOPHILIA.

United States Patent 3,652,530 ANTIHEMOPHHLIC FACTOR PREPARED FROM BLOODPLASMA USHJG POLYETHYLENE GLYCOL Alan .I. Johnson, New York, JackNewman, Pelham, and Margaret Howell Karpatkin, New York, N.Y., assignorsto American National Red Cross, Washington, DC. No Drawing. Filed Aug.28, 1967, Ser. No. 663,507 Int. Cl. A23j 1/06; C08h 1/00 U.S. Cl.260-]l12 B 11 Claims ABSTRACT OF THE DISCLOSURE Antihemophilic factor isseparated as a precipitate from fresh-frozen human plasma with a lowconcentration of ethanol, preferably 3 percent, at low temperature,followed by extraction with a low ionic strength buffer, preferably aTRIS buffer solution. The resulting product of intermediate purity(ZS-60X purified) can be lyophilized and stored or can be furthertreated to obtain a high purity AHF (ZOO-400x purified). High purity AHFis produced by further treating the intermediate purity product withPEG, preferably of a molecular weight of 4,000, in two steps, the firstat about 6 percent and the second at about 12 percent. The high purityproduct is lyophilized before storage. Both the intermediate, and thehigh, purity AHF are used in the treatment of classical hemophilia.

BACKGROUND OF THE INVENTION This invention relates to the separation ofblood proteins. More particularly, this invention relates to largescalepreparation of antihemophilic factor from human blood plasma.

It has been found that classical hemophilia is a deficiency diseasecaused by the absence of antihemophilic factor (AHF) in the blood. Ithas been further found that the AHF is concentrated in the blood plasma.Until recently, the only effective method of controlling bleeding inhemophiliacs was by the transfusion of fresh blood, fresh plasma orfresh-frozen plasma. The concentration of AHF in normal blood, or evenplasma, however, is so low that the patients circulation may beoverloaded before hemostasis can be achieved. Further, it is well knownthat AHF is susceptible to denaturation by heat, storage, freezing andthawing, thereby making the use of fresh whole blood or plasma extremelyundesirable. This has led to increased research in recent years towardextracting AHF from the plasma for storage and subsequent use.

The earliest attempts to isolate AHF from normal human plasma, forwidespread clinical use in hemophiliacs, were generally unsatisfactory.It was later found that fibrinogen prepared from human plasma by ethanolfractionation contained about X purified Other clinically useful AHFpreparations were produced by various modifications of the ethanolfractionation procedure. These preparations were more potent, being l015purified, and represented important therapeutic advance in hemophilia.Other methods were developed such as a recent method which utilizescryoprecipitation from single units of fresh-frozen plasma, and producesa preparation about 10-20 purified. Although the above procedures arecapable of being adapted to large scale use, the yield and purity of theproducts are relatively poor.

Thus, it can be seen that although considerable research has beenperformed in attempting to produce a commercially feasible high purityAHF preparation, there has been remarkably little success in thisregard. The methods used to separate AH F from human plasma on alaboratory scale either cannot be performed on a large scale or can beperformed only at prohibitive cost. In any event, these methods resultin a product which is comparatively low in purity, ranging from 5 toabout 20X purified. Other desirable characteristics of an AHFpreparation, which have not hitherto been achieved, are that theproducts should be stable, of known potency, easily soluble to a lowviscosity solution, and relatively inexpensive to produce.

It is, therefore, an object of this invention to provide a method offractionating blood plasma, and particularly to provide an AHFpreparation and a method for its production which is free of theaforementioned and other such disadvantages. More specifically, it is anobject of this invention to provide an inexpensive highly purified AHFpreparation of high stability and known potency.

It is yet another object of this invention to provide a method forseparating AHF from human plasma on a large scale. Another specificobject of this invention is to provide a relatively simple, inexpensivemethod for 0btaining AHF of intermediate purity from fresh-frozen bloodplasma by treatment with a low concentration of ethanol at a lowtemperature and subsequent extraction with a low ionic strength buffer.Still another specific object of this invention is to provide a methodfor obtaining highly purified AHF from fresh-frozen blood plasma bytreating the plasma with a low concentration of ethanol at lowtemperature, extracting with a low ionic strength buffer and furtherpurifying the product of intermediate purity with polyethylene glycol.

These and other objects of this invention will in part be obvious and inpart be pointed out as the description of the invention proceeds.

SUMMARY OF THE INVENTION Generally the inventive procedure utilizeslarge-scale ethanol cryoprecipitation of fresh-frozen human plasma andextraction of the AHF from the precipitate with tris- (hydroxymethyl)aminomethane buffer, hereinafter TRIS, to produce an intermediate purityAI-I F composition (25- 60 purified) followed by precipitation ofcontaminating proteins from the supernatant with polyethylene glycol,hereinafter PEG, resulting in a high purity AHF product (ZOO-400xpurified). Both the intermediate and high purity AHF may be used totreat hemophiliacs, as well as to prepare AI-LF of still higher purity.AHF purity, as used herein, refers to the concentration of AHF, in unitsper milligram of protein in the concentrate related to the AHFconcentration per milligram of protein in the starting plasma. Theinvention will be better understood by reference to the followingflow-diagrams in which Diagram I represents the procedure forpreparation of AHF of intermediate purity and Diagram II represents theprocedure for preparation of AHF of high purity.

pre-filter is used to remove the residual aluminum hydroxide. Whiledeprothrom-binization is preferable to insure against later untowardeffects, it has been theorized that this step may not be necessary, andshould not, therefore, be taken as unduly limiting. Trisodium citrate isadded as an anti-coagulant to a final concentration of 0.02 M to preventthe formation of minute amounts of thrombin which might labilize theAHF. This solution can be rendered sterile by Millipore filtration andthen lyophilized for a clinical product (intermediate purity), or usedas the starting material for further purification.

Purification by polyethylene glycol The TRIS extracted AHF supernatantis brought to about pH 6.0 by the gradual addition of 0.02 M citricacid. The lower pH results in a significantly higher purification. Theprotein concentration is then approximately 10-12 mgm./ml. At this pH, afinal concentration of 3 percent PEG-6,000 or 6 percent PEG-4,000precipitates most of the fibrinogen without removing the AHF. The PEGmay be added as a concentrated aqueous solution or as dry flakes. Theoperable concentration range of PEG-4,000 is about 3.5% to about 6.5%,with about 5.8% to about 6.0% being preferred. There is little increasein volume when dry flakes are used, which is a decided advantage withlarge-scale production. Vigorous mixing for at least 15 minutes,however, is required to insure complete solution of the dry PEG.Although the 6,000 molecular weight PEG may be used, we have found thePEG-4,000 is at least equally effective and has been tested extensivelyas a U.S.P. product: the latter is, therefore, preferred. The resultinglarge fiocculent precipitate is easily removed from the AHF-richsupernatant by centrifugation at 5,000- g. for 5 minutes at roomtemperature, and represents partially purified fibrinogen which may befractionated further.

Concentration of AHF by PEG The concentration of PEG in the supernatantis increased to 10 percent if PEG-6,000 or 12 percent if PEG- 4,000, toprecipitate the AHF and other residual proteins, and centrifuged for 10minutes at 6,000 g. The PEG- rich supernatant is discarded, most of theremaining PEG is removed from the walls of the centrifuged container andthe precipitate by 34 washes with ice cold 6 percent ethanol-TRISsolution, which selectively dissolves PEG, with agitation gentle enoughnot to dislodge the precipitate from the wall of the container. Theprecipitate may be redissolved in approximately 1,000th of the originalplasma volume with 0.02 M TRIS-0.02 M citrate bulfer at pH 7.0. Aftersterile filtration through a Millipore filter, the AHF solution may beshell frozen and lyophilized. For the final filtration of eitherintermediate or high purity AHF it is important to use a Millipore GSfilter which has a pore size of approximately 0.22 micron. It is alsopreferable to include an RA (approximately 1.2 microns) and an HA(approximately 0.45 micron) filter to prevent clogging of the GS bylarge molecular weight aggregates.

EXAMPLE I 211 packs of fresh-frozen human plasma weighing 45.6 kg. werewarmed for 5 minutes on each side at 50 C. The tare weight of the packswas 5.82 kg., the plasma being 39.78 kg., or a volume of 38.7851.

The plasma was then crushed and added to a container in a bath at to 1C. When half of the plasma was added, heating coils were placed into theice layer, liquid at 6 C. was circulated through the coils, and thebalance of the plasma was added. A liquid phase was allowed to form.

2 cc. of caprylic alcohol was added to reduce foaming. 2.311 1. of 53.3percent ethanol at --5 C. were added with gentle mixing over a period ofnot more than minutes using a sterile burette. Liquid was circulatedtherethrough at 6 C. The bath temperature was maintained at 0 to 1 C.The coils were not raised out of the ice layer, and the liquidcirculation to the bottom coils was stopped when only two inches offrozen plasma remained.

When the plasma had completely melted, the entire solution wastransferred into the pressure tank of a Sharples Centrifuge #2 andcentrifuged at a temperature of 0 to 1 C. using the /1 inch nozzle. Atthe start the small brine valve was fully opened, but was cut back A to/2 turn after the plasma surged out. The supernatant was discarded andthe precipitate, or paste, was extracted with 4.875 1. of 0.02 M TRIS(pH 7) at 25 C. The pH of the solution was checked and was 7.0. Thesolution was stirred gently with a Vibromixer, model E 1, set at 1%turns from off for 30 minutes and then centrifuged in cups at 25 C.,4500 r.p.m. for 10 minutes. The supernatant was decanted and theprecipitate was discarded. The supernatant was deprothrombinized at roomtemperature with 146.25 cc. of aluminum hydroxide. After the aluminumhydroxide was added the solution was stirred gently for 5 minutes andthen centrifuged at 4500 r.p.m. for 5 minutes at 25 C. The supernatantwas decanted and passed through a Millipore prefilter.

cc. of 0.5 M sodium citrate solution were then added and the solutionpoured into an 8 liter metal beaker using a 2 liter graduate to measurethe amount which was 5.055 1. 0.02 M citric acid was added using a 250cc. graduate to adjust the pH to 5.95-6.05, stirring continuously. Thevolume of citric acid solution added was 850 cc., bringing the totalvolume to 5,905 1.

Solid fla'lces of PEG 4,000 (CarboWax-4,000; Union Carbide Co.) weregradually added with the vibromixer in operation, to a finalconcentration of 5.8 percent, which required 343 grams. The mixing wascontinued for 15 minutes and then the solution was centrifuged for 5minutes at 4,500 r.p.m. The supernatant was decanted into an 8 litermetal beaker and the paste was discarded.

With the vibromixer running, PEG 4,000 was gradually added to the finalconcentration of 12 percent, or 366 grams. Mixing was continued for 15minutes and the solution was then centrifuged for 10 minutes at 4,000r.p.m. in several portions. Each time the supernatant was decanted anddiscarded and the precipitates were allowed to collect in the fourbottles of the centrifuge.

20 cc. of 0.02 M pH 7.0 TRIS-6 percent alcohol at 0 C. were added toeach bottle and swirled gently to flush out all excess PEG solutionwithout breaking up the precipitate. The wash solution was discarded,the mouth of each bottle was wiped with a gauze pad and the washingprocedure was repeated twice more.

The precipitate was dissolved in 195 cc. of 0.02 M TRIS- CI'FRATEsolution at 25 C. The diluent was added stepwise to each bottle andmixed gently until solution was complete. The pH was measured as 7.15.

The resulting solution was sterile filtered through a 142 mm. Milliporefilter press, using the prefilter and RA, HA, and GS filters, and filledinto 8 cc. amounts in vials. The vials of solution were then frozenrapidly in Dry Icealcohol and lyophilized. Both the intermediate andhigh purity AHF are extremely stable and have been kept at 37 C. forover three months.

EXAMPLE II Both the intermediate and the high purity AHF were assayedfor AHF activity and for impurities (Al+++ in the intermediate purityAHF, Al+++ and PEG in the high purity AHF).

AHF was assayed by a modified thromboplastin generation test asdescribed below. Serial dilution of a pooled normal human plasma controlas well as a lyophilized AHF concentrate were used as references. Eachdilution, made in 0.05 M imidazole bulfer at pH 7.3 was incubated in ageneration mixture consisting of the following:

0.2 ml. of dilution, 0.2 ml. of 1:10 dilution serum in imidazole salinebuffer (Celite activated), 0.2 ml. of deprothrombinized antihemophilicplasma diluted in imidazole buffer, 0.2 ml. of homolysate (prepared byfreezethawing washed human red cells 6 diluted 1:20-1:40 in normalsaline and 0.2 ml. of M/ 40 CaCl After 8-10 minutes incubation at 37 C.,0.1 ml. of this mixture was added along with 0.1 ml. of M/40 CaCl to 0.1ml. of pooled human plasma pre-incubated at 37 C. and the clotting timerecorded. The fastest clotting times obtained for each dilution wereplotted on the ordinate of log-log paper and the inverse of thedilutions on the abscissa, and joined by a straight line. Samples of AHFwere assayed in a similar way and a line of best fit drawn parallel tothe control line. The activity of the unknown was determined bycomparing the 15 second intercept dilution with that of the control.

Proteins were assayed by the biuret method (Colowick et al., Methods inEnzymology, Academic Press, N.Y., 3, p. 450, 1957) and protein nitrogenby gas chromatography.

The intermediate purity material had a specific activity of 0.2-0.4units/mg. protein, and the high purity product had a specific activityof 36 units/mg. protein. 1 ml. of frozen pooled normal plasma usuallycontains 0.8 unit of AHF.

Aluminum assays of the intermediate purity AHF are shown in Table I.

TABLE 1 pg. Al lunit intermediate purity AHF #1;- #2.- AF' Lot No unitAHF Lot No. unit AHF N or1-:.l\1can=0.1116. Standard deviation=5.585

The PEG assay was performed by a modification of the method of Shatteret al. (Renal excretion and volume distribution of some polyethyleneglycols in the dog, Anal. Chem., 19 p. 32, 1947). The PEG assay of thehigh purity AHF can be seen in Table II.

TABLE 11 PEG content of high purity AHF AHF activity, PEG, Mg: PEG] LotNo rug/unit mg./ml. unit AHF Mean 61. 87 2. 407 0. 056 Standarddeviation. 44. 58 1.500 0. 04407 EXAMPLE III Twenty-seven patients havereceived intermediate purity AHF concentrate prepared by the ethanolcryoprecipitation-TRIS method, 23 with classical hemophilia and 4 withvon Willebrands disease, during 29 hospital admissions. Three of thesehemophiliacs took part in studies to estimate the in vivo recovery ofAHF, and the other 24 were treated for spontaneous or traumatichemorrhage or to ensure hemostasis during and after surgery.

The following report on these patients is given in Table III. Theirhemostatic response to the drug was excellent. Apart from Patient G. C.,who had a demonstrable AHF inhibitor, and Patient B. B., in whom nonewas demonstrated, the concentrate maintained satisfactory AHF levels 25%of normal following surgery or severe trauma and 15 of normal followingminor bleeding episodes), with normal hemostasis.

Two patients complained of circumoral tingling during the actualinfusion but it stopped immediately afterward. Their vital signs wereunaltered. The temperature in one patient (R. W.) was elevated afterinfusion of one batch of AHF but other batches had no such efiect.Hemolysis, observed in one patient (T. D.), might have been due to verylow levels of anti-A blood group antibodies present in the preparation.Antibody proteins have a relatively long half life in the circulation(variously reported as 14 to 30 days), and there may be a cumulativeeffect during prolonged treatment. This complication was also reportedwith AHF concentrates prepared by other methods.

No other untoward reactions were noted even in patients who habituallyhad had severe allergic reactions to freshfrozen plasma.

TABLE 111 Summary of clinical data on intermediate-purity AHF preparedaccording to the ethanol cryoprecipitation-TRIB method Highest Unitsplasma AHF AHF level Duration of adminrecorded, Patient Diagnosistreatment istered 2 percent T.S Severe AHF deficiency, recovery study.30 min. approx.-. 3,150 65. W.C do d0 3,700 90. .d0 do 3,150 55. V.

1st admission. Severe AHF deficiency, eraniotomy and evacuation ofsubdural hemorrhage 200: 2d admission..- Traumatic cerebral hemorrhage68. 111.0 Severe AHF deficiency, post-traumatic subdural hern0rrhage 71.\V.D Severe lien1ophMia-hemorrhage into spinal cord 10 days. .000 153. N.M Moderate hemophilia (12% AHF), thoracetoruy and biopsy of mediastinalmass and 12 days 30,250 Unknown.

pericardium.

TABLE III.Con tinued Highest Units plasma AHF AHF level Duration ofadminrecorded, Patient Diagnosis treatment istered percent 1.0 Severehemophilia, reduction and repair of strangulated hernia 8 days 13, 80050. LR Severe hemophilia, spontaneous intraabdominal and intrathoi'aciehemorrhage, epis- 14 days 34, 118 Unknown.

taxis, GI bleeding. A.R Severe hemophilia, GI bleeding 9 days 20,000 Do.ILA Gangrenous bowel 3 weeks approx 3 48, 000 100. 1.D Removal of medialmeniscus from knee 2 weeks 49,160 100. B.B Severe hemophilia, fracturedleg- 3 days- 7,590 1. E.F Severe hemophilia, torn knee ligaments 8 day25,000 Unknown. J11 Severe hemophilia, lip laceration; previousadministration of sufiicient FFP to cause 7 days 9,200 Do.

pulmonary congestion failed to stop bleeding. W.l" Severe hemophilia,extraction of 8 permanent teeth ..do 9, 000 35. G.O Two-year oldhemophiliac with AHF inhibitor, bleeding from cutdown site 4 days- 4,984 1. AJ Severe hemophilia, hematuria; no response to previousadministration of FFI and 1 infusion 2,013 106.

cryopp G.F.:

1st admission... Severe hemophilia, hematomas of buttocks 24 hours 2dadmission.... Hematomas of arm and hand 1 infusion W.A Severehemophilia, hemorrhage into forearm following vein-puncture. ..do. D.W-Extraction of teeth 4 days. S.R ..do--. J .M 7 days L.M. Recoveryexperiment 1 infusion S.C. Extraction of teeth 18 hours B.U. (von W.)Traumatic hemarthrosis of shoulder 1 infusion J.H. (von W.) G.I.bleeding do 1 Plasma AHF level: 1%. 3 Approximately. 1 1 unit AHF=AHFactivity in 1.0 ml. of fresh pooled normal plasma. 4 Von Willebrandsdisease.

EXAMPLE IV thereafter. The total volume of the concentrate injectedNineteen patients with classical hemophilia have reper day ranged from16 to 72 (equlvalent ceived the high purity AHF concentrate prepared bythe 10004700 P of'plasma); After the fiIst post'operatlve PEG method: 11participated in studies to estimate in f y a Smgl? lnlectlon Was glvenevery 12 hoursp vivo recovery after intravenous injection and 2particinon, bleedlng was Comparable to that Observed 111 normal pated inintramuscular injection studies. The concentrate Chlldfen, I10POSt-OPBIQUVG bleedlng Occurred, and rate was administered to the othersix to ensure hemostasis of healing was normal. No toxic reactions wereencounduring and after surgery; they were first given this AHF tered.for Pefiods p to one Week, and lmefmedlate p y mate- When the AHF wasinjected intravenously into volunrial vgas substituted for the remainderof the treatment tears with severe hemophilia, approximately 70 percentperio of the original dose was recovered. AHF levels exceeding Thefollowmg report on the 18 patlents of a 30 percent (adequate to preventbleeding due to severe marized table. The hemostatic response tointravenous injection of the concentrate was excellent: satisfactory faccldenta} were obtalned after AHF level was maintained 25% f normal),blood single 10-30 ml. in ection. Little AHF was recovered 1n loss andwound healing were normal. the plasma, however, after intramuscularinjection.

TABLE IV Summary of clinical data on high-purity AHF prepared accordingto the PEG method Highest Units plasma AHF AHF level Duration ofadminrecorded, Patient Diagnosis treatment istered 1 percent 'I.D SevereAHF deficiency, dental surgery, repair of herniadays 91 GN Severe AHFdeficiency, excision of pilonidal sinus 7 days. ,000 150 G.F- Severe AHFdeficiency, I.M. recovery study 1 minute 1,080 1 W.A ..do ..do 4,800 1 11 unit AHF=AHF activity in 1.0 ml. of fresh pooled normal plasma. 2 Thismaterial of intermediate purity.

It can be seen that several of the patients were children Thus, it canbe seen that the method of this invention, wi h severe h p whose agesranged from 7 as exemplified by the preferred embodiment but not Whentreated with the AHF of the instant invention they i i thereto, can beused efficienfly to prepare a human ai ed hgmostaisis fluring g after gi AHF concentrate of intermediate or high purity which is r c onso rimaran erman n ee 22 s ig g g l performed on clinically useful and extremelystable in storage. Certain each patient. Plasma AHF levels above 25percent were modlficatlons and embodlments can be made to thlsmaintained during the operative period and for a week vention withoutdeparting from the spirit or scope thereof.

What is claimed is:

1. A method of preparing highly purified AHF comprising the steps of:

(a) mixing plasma with from about 2 percent to about 4 percent by volumeof ethanol at a temperature between about and about +4 C. to obtain aprecipitate containing AHF;

(b) extracting said precipitate with a low ionic strength butlersolution comprising tris (hydroxymethyl) aminomethane at roomtemperature to obtain a solution comprising AHF and said buffersolution;

(0) deprothrombinizing said solution with aluminum hydroxide gel;

(d) buffering said solution to a pH of about 5.6 to

about 7.0;

(e) adding polyethylene glycol to said solution to a concentration offrom about 3.0 percent to about 6.5 percent to obtain a supernatantsolution and a precipitate;

(f) adding polyethylene glycol to said supernatant to a concentration offrom about 10 to about 12 percent to obtain a precipitate including AHF;and,

(g) recovering AHF of from about 200 to about 400x purity.

2. The method of claim 1, wherein said plasma is mixed with about 3percent by volume of ethanol.

3. The method of claim 1, wherein said low ionic strength buffersolution has a concentration of from about 0.005 to about 0.02 M and apH of about 7.0.

4. The method of claim 1, wherein said solution is buffered in step (d)to a pH of from about 5.8 to about 6.4.

5. The method of claim 4, wherein said solution is buffered in step (d)to a pH of about 6.0.

6. The method of claim 1, wherein said solution is buffered in step (d)with a buffer comprising sodium citrate and citric acid.

7. The method of claim 1, wherein said polyethylene glycol is added instep (e) to a concentration of about 5.8 percent.

8. The method of claim 1, wherein said precipitate from step (f) isdissolved in 0.02 M tris (hydroxymethyl) aminomethane-citrate solutionand sterile filtered through a filter having a pore size of about 0.22microns before recovering said AHF.

9. The method of claim 1, wherein step (g) is performed bylyophilization.

10. Highly purified AHF prepared by the method of claim 1 utilizinghuman blood plasma as the starting material.

11. A composition consisting essentially of highly purified AHF preparedby the method of claim 1 in aqueous solution utilizing human bloodplasma as the starting material.

References Cited UNITED STATES PATENTS 2,390,074 12/1945 Cohn 260-1222,867,567 1/1959 Bidwell 424-177 3,415,804 12/1968 Polson 2601 12FOREIGN PATENTS 883,549 11/1961 Great Britain 16774 H OTHER REFERENCESNature, vol. 203, 1964, p. 312, Pool et al. The Proteins, vol. III,1965, p. 372, Neurath. Blood, vol. 28, p. 1011, Johnson et al., December1966.

HOWARD E. SCHAIN, Primary Examiner US. Cl. X.R. 424-101, 177

